Papillomaviruses are now understood to be very important causal agents in human disease. As part of their normal life cycle, these viruses infect and transform cells in the epithelium causing benign tumors that with a low but significant frequency can become malignant. A deeper understanding of the life cycle in general, and DNA replication in particular, is of critical importance for the understanding of the disease, its transmission and ultimately for the development of effective therapeutic measures. Although recently developed vaccines promise to reduce the prevalence of this disease, for the foreseeable future papillomavirus infections will still present a formidable problem due to the large number of already infected individuals. Thus treatment options such as small molecule inhibitors of the viral life cycle would be desirable. Compared to other disease causing viruses very limited effort has been invested in identifying targets for small molecule intervention in papillomaviruses. Our goal has for many years been to establish a sufficiently detailed understanding of the papillomavirus DNA replication apparatus to identify molecular interactions that could serve as targets for small molecule intervention. Work by us and others has now reached the point where we can pinpoint particular aspects of the viral replication machinery that represent plausible targets for small molecule interference with viral replication. In Specifi Aim 1, we propose to characterize 4 intra-molecular interactions in the papillomavirus E1 initiator protein that constitute such potential targets. We will analyze these interactions biochemically and genetically to determine their specific functions, their degree of conservation within the papillomavirus family, and whether they are essential in the viral life cycle. An interesting aspect of these intra- molecular interactions is that they are controlled by phosphorylation by the protein kinase CK2. In Specific Aim 2, we will determine the role that phosphorylation of the viral E1 and E2 proteins play in the control of viral DNA replication. Our results show that phosphorylation of these two proteins result in dramatically altered biochemical activities including loss of DNA binding activity. We will investigate the mechanism by which the phosphorylation events cause biochemical changes in the E1 and E2 and what function these phosphorylation events serve in the viral life cycle. These studies will result in a greatly improved understanding of how papillomaviruses regulate initiation of DNA replication. They will also provide information about whether intra- molecular interactions in E1 can be developed into targets for small molecule intervention in papillomavirus infections.